The envelope of Gram-negative bacteria consists of two membranes separated by the periplasmic compartment that contains the peptidoglycan wall. The inner membrane (IM) is in contact with the cytosol while the outer membrane (OM) contacts the extracellular environment. The OM is a unique structure, essential for Gram-negative bacteria, composed of lipopolysaccharide (LPS), phospholipids and proteins. It is a very selective permeability barrier that allows the bacteria to survive in hostile environments such as the gut, where the OM resistance to bile salts allows enteric bacteria to thrive. The components of the OM are the first to come in contact with a host upon infection and strongly modulate the interaction of symbiotic and pathogenic bacteria with their host. A clear understanding of the OM biogenesis process is essential to understand host? pathogen interactions as well as a fundamental aspect of bacterial physiology. Outer membrane proteins (OMPs) are integral membrane proteins with b-barrel structures embedded in the OM. Many OMPs are immunogenic and some of them serve as adhesins mediating adhesion and colonization of host tissues. OMPs are synthesized in the cytosol and translocated across the IM by the Sec translocation machinery and inserted specifically in the outer membrane by a multiprotein complex known as b-Barrel Assembly Machine (BAM). However, the molecular mechanisms by which OMPs are targeted to the OM and inserted by BAM are poorly understood. In this proposal, we will define the fundamentals of OMP transport and assembly focusing on the BAM complex. We will (i) define the mechanism of OMP targeting to the OM and (ii) test mechanistic hypotheses of OMP insertion derived from the high- resolution structures of BAM.